The essential roles of the ubiquitin-proteasome system (UPS) in many cellular processes are now well recognized. However, many questions still remain unanswered. Intriguing yet not fully understood are the functions of the immunoproteasome, an alternative form of the constitutive proteasome. It has been shown that the roles of the immunoproteasome in cells are not just limited to the generation of antigenic peptides, but far more multifaceted. In this regard, recent studies have shown that catalytic subunits of the immunoproteasome are implicated in various types of diseases including cancer. Despite these therapeutic implications, the immunoproteasome remains largely untapped for cancer drug discovery. A better understanding of immunoproteasome functions would be crucial in developing the optimum LMP2 inhibitor(s). In our continuing efforts to better understand the role of the immunoproteasome in cells, we have recently developed a small molecule probe ('UK-101') that specifically targets LMP2, a major catalytic subunit of the immunoproteasome. Further, we found that LMP2 is highly expressed in several types of primary tumor tissues and cancer cell lines. Interestingly, cancer cells that highly express LMP2 are markedly more sensitive to UK-101 than those expressing low level of LMP2. Consistent with these in vitro results, UK-101 inhibits tumor growth in a mouse xenograft model of human prostate tumor that highly expresses LMP2, but not that of tumor that expresses low level of LMP2. These observations further support a potential role of LMP2 in cancer cell growth and survival. The goals of this application are to determine the roles of the immunoproteasome in prostate cancer and to investigate therapeutic potentials of the immunoproteasome-targeting approaches in cancer therapy. Towards this goal, we propose to use innovative chemical probes that selectively target and visualize individual catalytic subunits of the immunoproteasome. In order to accomplish this goal, four specific aims are proposed: 1) To validate the immunoproteasome as the major pharmacological target of UK-101, 2) To determine dynamic cellular distribution of the catalytically active immunoproteasome, and 3) To determine the unique functions of the immunoproteasome compared to the constitutive proteasome and 4) To develop an optimum LMP2 inhibitor(s) by generating a structurally diversified small molecule library. The proposed work integrates both chemical and biological approaches. Successful completion of the project will significantly expand our knowledge on the role of the immunoproteasome. In addition, the study will yield critical information in developing immunoproteasome-specific inhibitors as a potential therapeutic agent. Finally, small molecule probes that will be used in this application will provide valuable research tools for the ubiquitin-proteasome biology community.